Samples Of Biological Material Research Articles

Changing natural conditions and their impact on the Mt. Śnieżnica landscape, Outer Western Carpathians – Reconstruction of the Holocene environment based on geochemical indices and radiocarbon dating

Environmental changes during the Holocene impacted the development of all civilizations, and it is important to understand the power of this influence through, for instance, the reconstruction of these changes. However, when the climate and environmental conditions of the deep past are analyzed, researchers need to rely on various types of proxy data that are only approximations of the required information. In addition, this type of information is often absent or has several gaps (hiatuses). In the present study, we analyzed a 4.4-m deep core excavated from the fen formed within the landslide body on the northern side of Mt. Śnieżnica in the Wyspowy Beskidy Mountains, the Outer Western Carpathians, southern Poland. In total, we analyzed 405 samples in terms of 29 geochemical components (e.g., nitrogen (N), carbon (C), sulfur (S), and the total organic carbon (TOC)) and physical properties, namely particle-size distribution, loss on ignition (LOI), and microcharcoal content. Additionally, to establish geochronology, we dated 27 samples of different biological materials using the Accelerator Mass Spectrometry radiocarbon method. A detailed examination of plant macrodetritus and wood anatomy supported our interpretation based on the geochemical data. The Mt. Śnieżnica landslide probably formed ca. 14,000 cal BP in the first phase of the Allerød Interstadial. For almost 9000 years, there were no appropriate terrain conditions for the long-term accumulation of organo-mineral materials. At ca. 4400 cal BP, peat accumulation commenced. The beginning of peat accumulation correlates with the global 4.2 Bond event of cold climate conditions. After another ca. 2000 years, the core sediments were dominated by limnetic mud, suggesting aquatic conditions in the landslide depression. This sudden shift in the characteristics of sedimentation is loosely linked to the boundary between the Subboreal and Subatlantic phases (ca. 2500 cal BP). The apparent dichotomy of the depositional record agrees with the reconstructed climatic conditions during the second part of the Holocene. Up to 3000 cal BP, the regional climate was warm and humid, which allowed fast biomass production and hillslope stabilization by trees. Forest fires occurred only at the beginning and end of this period (4400–3000 cal BP). After 3000 cal BP, the regional climate became cool and dry. In this period, we found evidence of intensified erosion, but it was unrelated to forest fire activity.

Epizootological analysis of the prevalence of salmonellosis in poultry in Ukraine in 2012–2021

The main infectious agent that causes contamination of poultry products are bacteria of the genus Salmonella. The article presents the results of monitoring studies on salmonellosis among poultry of different technological direction of poultry farms of all forms of ownership in Ukraine. In the analysis of the results of the incidence of salmonellosis in poultry used Reports on the work of state laboratories of veterinary medicine of the State Food and Consumer Services of Ukraine for 2012–2021. Data from the State Statistics Service of Ukraine were also used during the work on the article. It was found that for the period 2012–2021, the total number of samples tested for salmonellosis was 306466, of which 932 were positive, which was 0.3 %. It is established that the number of studies in certain years differs greatly in number. After all, the peak number of studies fell on 2012 (51985 samples), but in 2020 it was only 25.796 samples or 49.6 % to the peak level of studies. The highest percentage of pathogen isolation was observed in the period from 2013 to 2015, respectively 0.39, 0.48 % and 0.38 %. In 2016–2018, there was a decrease in the number of positive samples, respectively, the number of positive samples was 0.24–0.33 %, the lowest percentage in 2019–2021, the number of positive samples was minimal and amounted to 0.05 %, 0.22 % and 0.13 %. Thus, there is a clear trend of decreasing the amount of pathogen. The largest number of positive samples of biological material for salmonellosis in the period 2012–2021 was found in Sumy and Luhansk regions, 156 and 186 samples, respectively. Slightly fewer positive samples were found in Kharkiv (117), Kirovohrad (79), Cherkasy (71), Zaporizhia (51), Donetsk (45), Kyiv (30) and Zhytomyr (30) regions. The pathogen Salmonella spp. in Zakarpattia, Rivne and Chernivtsi regions.

Epidemiological situation of Avian Influenza in Ukraine during 2020–2021

Avian influenza viruses (AIVs) are spread globally by wild migratory birds that are reservoirs of AIVs. Epidemics of highly pathogenic avian influenza (HPAI) can devastate the poultry industry and result in severe trade restrictions. Many routes of wild migratory birds pass through the territory of Ukraine, therefore studying the circulation of the viruses is important for the prevention of AIV spreading. The aim of the investigation was to summarize and analyze results of AI tests carried out by state laboratories during 2020–2021. The tests were conducted in the framework of state control of AI aimed to early detection and prevention of HPAI virus spreading on the territory of Ukraine. 27 354 samples of biological material from wild and domestic birds (11 115 samples – in 2020 and 16 239 – in 2021) were tested by PCR in State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise and regional state laboratories. For PCR testing commercial kits and reagents were used: IndiSpin Pathogen Kit (INDICAL BIOSCIENCE), VetMAX™-Gold AIV Detection Kit (Thermo Fisher Scientific), AIV H5-H7 REAL TIME (ADIAVET), AgPath-ID™ One-Step RT-PCR Reagents (Ambion) with N8 RT-PCR primers and probe. In 2020, 84 positive samples on AI type A subtype H5N8 from poultry of Vinnytsia, Kherson, Kyiv and Mykolaiv oblasts was detected. In 2021, the presence of AIV RNA subtype H5N8 and H5N1 was detected in 87 samples (72 samples from poultry, 11 from wild birds and 4 from zoo birds) of Mykolaiv, Kyiv, Donetsk, Ternopil and Kherson oblasts. In 2020, 9 outbreaks of AI were registered in Vinnytsia (1), Mykolaiv(5), Kyiv (2) and Kherson (1) oblasts; in 2021, 11 outbreaks were registered in Mykolaiv (4), Kyiv (2), Donetsk (2), Ternopil (1) and Kherson (2) oblasts. In general, during 2020–2021, 20 outbreaks of AI subtype H5 (3 – commercial poultry farms, 13 – from backyards, 3 – in wild birds and 1 – in zoo birds) were registered in 6 oblasts of Ukraine. RNA of AIV in the samples from imported birds was not detected. Wild migratory birds are the most likely source of AIV in Ukraine since most outbreaks were registered in oblasts located at the crossing of wild bird migratory routes.

Antibiotic resistance of enterobacterial pathogens isolated on the territory of the Northern Kazakhstan

The purpose of this study was to isolate and identify strains of infectious disease pathogens and to determine their antibacterial drug resistance. From 2018–2020, 5322 samples of biological material from animals and birds, products of animal origin were taken at retail outlets, livestock enterprises in Kostanay and North Kazakhstan regions. In the study territories of 20 districts and 4 cities were covered. Phenotypic resistance analysis of the recovered isolates of Salmonella spp. showed a high level of resistance to antibiotics of the tetracyclines (82/137) and nitrofurans (81/137) groups. S. aureus isolates had increased resistance to the β-lactams (124/131) and macrolides (96/131) groups. As a result, the largest number of genes encoding resistance was found to the β-lactams, aminoglycosides, tetracyclines and macrolides groups. The study was carried out within the framework of the project AR05131447 “Monitoring of antibiotic resistance of pathogens of enteropathogenic zooanthroponosis diseases in the Northern Kazakhstan”.

Urogenital infections of women of reproductive age caused by conditionally pathogenic microorganisms on the background of tobacco smoking

The aim: to study changes in the microbiota of the urogenital tract of women of reproductive age in the Dnipro region, which is caused by conditionally pathogenic microorganisms on the background of smoking.
 Material and methods: biomaterial of patients (scraping from the urogenital tract) who applied to the Center for Laboratory Medicine PE “VIS-MEDIC” in Dnipro region. We analyzed the components of the microbiota of the vagina of women of the surveyed groups (sign of age and smoking) in the period from 2018 to 2021. For the use in an analysis, microbiota data were obtained using the test system Femoflor Screen.
 Results. Analysis of the results of the study revealed the dependence of the composition of the microbiota of the reproductive tract of women on the use of tobacco products. An increase in indicators for conditionally pathogenic microorganisms was shown. The rate of detection of elevated levels of M. hominis in samples of biological material varied between 6-8 % but was not recorded in all study groups of women. There was also an increase in the frequency of detection of elevated levels of U. urealyticum, U. parvum in samples of biological material, the values of which ranged from 9 to 50 %.
 Conclusions. The obtained data allowed to assess the effect of tobacco on the composition of the microbiota of the urogenital tract of women and made it possible to use them in measures of social and preventive work, as an indisputable fact to quit smoking. Therefore, there is a need for further research to establish the role of microorganisms involved in restoring the composition of the microbiota after inflammatory processes in women who use and do not use tobacco products.
 The results may be relevant for the diagnosis of inflammatory diseases, processes caused by opportunistic pathogens of the urogenital tract of women of reproductive age, potentially dangerous occurrence and development of infertility and the basis for social and preventive work among women on the background of smoking

A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene.

The nucleotide sequence of the BRAF codons 592-602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799-1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801-1793). The analytical characteristics of the proposed methods for somatic mutations' detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules. The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9-50.0%. A complex of methods for determination of the nucleotide sequence of the BRAF codons 592-601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600-601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

FEATURES OF BIOFILM FORMATION OF SOME PATHOGENIC AND COMMENSAL ESCHERICHIA COLI ISOLATED FROM THE BODY OF DOGS AND CATS

The relevance of this study is conditioned upon epidemic growth of nosocomial infections, which include Escherichia Coli (E. coli). One of the factors of pathogenicity of such microorganisms is the ability to form a biofilm – a complex community, within which bacteria acquire increased resistance to environmental factors, primarily to antibacterial drugs, which considerably complicates the course of the infectious process. In this regard, the purpose of this study was to determine the features of the formation and dependence of the density of the formed biofilm on the antibiotic resistance of pathogenic and commensal E. coli strains isolated from dogs and cats. The resistance of E. coli isolates to antibacterial drugs was established according to the disk diffusion method, according to EUCAST recommendations. The ability of microorganisms to form biofilms and determine their density was investigated in sterile plastic 96-well plates. The ability to form biofilms was assessed visually and microscopically, the density of biofilms was determined in units, spectrophotometrically, by the optical density of the washing solution. The paper presents the results of a study of 63 samples of pathological (wound infections) and biological material. From them, 10 E. coli isolates were obtained (6 from dogs and 4 from cats), which were selected for further research. It was established that all E. coli isolates had the ability to form phenotypic biofilm. The study investigated the interdependence of antibiotic resistance of E. coli isolates and their ability to form biofilms. Thus, isolates that were parted from pathological material and had a positive reaction on the CHROMagar™ ESBL medium for the determination of extended-spectrum beta-lactamases had greater resistance to various groups of antibacterial drugs and formed high- and medium-density biofilms, while E. coli isolates parted from pathological and biological materials with a negative reaction on CHROMagar™ ESBL medium formed a low-density biofilm and had less resistance to different groups of antibacterial drugs. The results obtained allow searching for innovative, sometimes alternative, methods of treatment and prevention of pathologies caused by them Keywords: microorganism, biofilm, antibiotic resistance, antibacterial drugs, wound infection

Stokes Dynamic Polarimeter for Non-Organic and Organic Samples Characterization.

The light polarization properties provide relevant information about linear–optical media quality and condition. The Stokes–Mueller formalism is commonly used to represent the polarization properties of the incident light over sample tests. Currently, different Stokes Polarimeters are mainly defined by resolution, acquisition rate, and light to carry out accurate and fast measurements. This work presents the implementation of an automatic Stokes dynamic polarimeter to characterize non-biological and biological material samples. The proposed system is configured to work in the He-Ne laser beam’s reflection or transmission mode to calculate the Mueller matrix. The instrumentation stage includes two asynchronous photoelastic modulators, two nano-stepper motors, and an acquisition data card at 2% of accuracy. The Mueller matrix is numerically calculated by software using the 36 measures method without requiring image processing. Experiments show the efficiency of the proposed optical array to calculate the Mueller matrix in reflection and transmission mode for different samples. The mean squared error is calculated for each element of the obtained matrix using referenced values of the air and a mirror. A comparison with similar works in the literature validates the proposed optical array.

Assessment of the Application of Erythrocytal Diagnosticum (Lyophilizate) in Detecting Tularemia Agent in Natural Foci

Tularemia is a zoonotic disease with a wide geographical dissemination, and its causative agent Francisella tularensis can be used as a bioterrorism agent. The aim of the study was to evaluate the use of a set of reagents “Erythrocytic immunoglobulin dry tularemia diagnosticum” (“DET-Ig”) with the help of control test strains and field material from natural tularemia foci. Materials and methods. Using the introduced erythrocyte diagnosticum, we studied the decontaminated cultures of test strains (F. tularensis Miura, F. tularensis 55, F. tularensis Schu, F. tularensis 15 NIIEG, Brucella abortus 544, B. melitensis 16-M, B. suis 1330, and Yersinia enterocolitica 64, Y. enterocolitica 178, Y. enterocolitica 383) and environmental samples suspected of containing F. tularensis. Results and discussion. It has been proven that the developed diagnosticum is specific, sensitive, and easy to use for routine diagnostics of tularemia. In the course of laboratory tests of the experimental series of the DET-Ig reagent kit, the possibility of qualitative determination of the tularemia agent in bacterial cultures, biological material and environmental samples in the reaction of indirect hemagglutination was demonstrated. Comparison of the results of use of erythrocyte diagnosticum in liquid and lyophilized forms showed the advantages of drugs after lyophilization: the possibility of transportation and long-term storage at any temperature conditions in various climatic conditions; the setting of the reaction is possible without the use of special diluents. The guaranteed storage term is set for two years (observation period). The results obtained indicate the prospects of introducing the developed drug into healthcare practice.

Development of multi-frequency impedance scanning electron microscopy.

Nanometre-scale observation of specimens in water is indispensable in many scientific fields like biology, chemistry, material science and nanotechnology. Scanning electron microscopy (SEM) allows high-resolution images of biological samples to be obtained under high vacuum conditions but requires specific sample-preparation protocols. Therefore, there is a need for convenient and minimally invasive methods of observing samples in solution. We have developed a new type of impedance microscopy, namely multi-frequency impedance SEM (IP-SEM), which allows nanoscale imaging of various specimens in water while minimising radiation damage. By varying the frequency of the input voltage signal of the sine wave, the present system can detect dielectric properties of the sample’s composition at nanometre resolution. It also enables examination of unstained biological specimens and material samples in water. Furthermore, it can be used for diverse samples in liquids across a broad range of scientific subjects such as nanoparticles, nanotubes and organic and catalytic materials.

The German Uranium Miners’ Biobank—A Biobank for OMICs Radiation Research

Systematic bio- and databanks are key prerequisites for modern radiation research to investigate radiation response mechanisms in the context of genetic, environmental and lifestyle-associated factors. This report presents the current status of the German Uranium Miners’ Biobank. In 2008, the bio- and databank was established at the Federal Office for Radiation Protection, and the sampling of biological materials from former uranium miners with and without lung cancer was initiated. For this purpose, various biological specimens, such as DNA and RNA, were isolated from blood samples as well as from formalin-fixed paraffin-embedded lung tissue. High-quality biomaterials suitable for OMICs research and the associated data on occupational radiation and dust exposure, and medical and lifestyle data from over 1000 individuals have been stored so far. Various experimental data, e.g., genome-wide SNPs, whole genome transcriptomic and miRNA data, as well as individual chromosomal aberration data from subgroups of biobank samples, are already available upon request for in-depth research on radiation-induced long-term effects, individual radiation susceptibility to lung cancer and radon-induced fingerprints in lung cancer. This biobank is the first systematic uranium miners´ biobank worldwide that is suitable for OMICs research on radiation-exposed workers. It offers the opportunity to link radiation-induced perturbations of biological pathways or processes and putative adverse outcome(s) by OMICs profiling at different biological organization levels.

Potential for the use of cards for sampling and transportation of biological material during forensic chemical and toxicological examinations

The objective of the study is to evaluate the potential for the use of cards for sampling and transportation of biological material during forensic chemical and toxicological examinations by the example of a biological sample, urine containing zopiclone. Two methods of sample preparation were compared. The use of cards for the collection and transportation of biological material, such as urine, followed by high-resolution high-performance liquid chromatography-mass spectrometry (HPLC-MS) for the identification of zopiclone metabolites was shown to be beneficial in forensic chemical and toxicological examination. The validation evaluation of the proposed sample preparation and identification method met the acceptance criteria.

Cell Adhesion and Biofilm Formation Analysis.

Cell adhesion to surfaces and ulterior biofilm formation are critical processes in microbial development since living in biofilms is the preferred way of life within microorganisms. These processes are known to influence not only microorganisms development in the environment, but also their participation in biotechnological processes and have been the focus of intense research that as a matter of fact, was mainly directed to the bacterial domain. Archaea also adhere to surfaces and have been shown forming biofilms, but studies performed until present did not exploit the diversity of methods probed to be useful along bacterial biofilm research.An experimental setup is described here with the aim of stimulating archaeal biofilm research. It can be used for studying cell adhesion and biofilm formation under controlled flow conditions and allows performing in situ optical microscopy (phase contrast, fluorescence, or confocal) and/or spectroscopic techniques (UV-Vis, IR, or Raman) to determine structural and functional biofilm features and their evolution in time. Variants are described with specific aims as working in anaerobiosis and allow sampling of biological material along time.

Analysis of Mycoplasma pneumoniae antibiotic resistance based on data obtained in the Tula region

Objective. To study the prevalence of mutations causing resistance to macrolides in Mycoplasma pneumoniae in the Tula region and compare the data with similar studies from European and Asian countries. Material and methods. A retrospective study of 76 samples of biological materials (nasal and pharynx swabs) was conducted. The materials were collected from the patients with an established diagnosis: the lower respiratory tract infection (pneumonia, bronchitis). All the patients included in the study were treated in specialized infections units and clinics of the Tula region for the period 2017–2018. Antibiotic therapy was not conducted before the initial survey. All the samples contained M. pneumoniae DNA. A real-time polymerase chain reaction (real-time PCR) technology was used to detect the DNA of M. pneumoniae in the materials. The analysis of mutations in the 23S rRNA gene was performed at the Scientific Research Institute of Antimicrobial Chemotherapy in Smolensk. During the study, a modified real-time PCR technique with the effect of the fluorescence quenching of the probe with a primer (patent No. 2646123) was used. The statistical analysis was carried out with the MS Excel software package. Results. It was identified that 9 samples (11.84%) had mutations associated with resistance to macrolides. Two samples had the A2059G mutation, while seven samples had the A2058G mutation (numbering of nucleotide sequences in the M. pneumoniae rRNA gene was compiled following E. coli numbering). The data obtained by us are slightly higher than the mean values of European countries and are significantly lower according to the studies of Asian countries. Conclusions. Provided unique data on the prevalence and identification of mutations of macrolide-resistant strains of M. pneumoniae in one of the regions of the Central Federal District is a contribution to the study of antibiotic-resistant strains of M. pneumoniae in Russia and all over the world. There is a necessity to monitor antibiotic resistance in other regions and increase diligence on atypical forms of community-acquired pneumonia since the co-infection of M. pneumoniae patients with COVID-19 leads to a deterioration in the condition and an increase in the duration of treatment. Keywords: community-acquired pneumonia, macrolides mycoplasmas, mutations of antibiotic resistance

Интенсивность процессов окислительной модификации белков женского и коровьего молока

Breast milk is a source of all the essential nutritional components necessary for the full growth and development of the child, therefore, it is necessary to study its composition and physical and chemical properties in order to adapt human milk substitutes. Adapted infant milk formulas are produced mainly from cow's milk, bringing formula nutrient composition closer to the composition of women's milk, adapting it in accordance with the requirements of the infant body. However, technological processes for the production of dairy products contribute to the activation of oxidative reactions, the violation of protein conformation. The purpose of the study was to compare the intensity of formation of carbonyl derivatives of human and cow's milk proteins during spontaneous and metal-catalyzed oxidation. Material and methods. The object of the study were samples of mature milk of healthy nursing mothers (n=12), and samples of drinking ultra-pasteurized milk for baby nutrition (n=8) which were used as a comparison material. The intensity of oxidative modification of milk proteins was determined spectrophotometrically by the interaction of carbonyl derivatives of amino acid residues with 2.4-dinitrophenylhydrazine to form 2.4-dinitrophenylhydrazone derivatives in a native sample of biological material and under induction of protein oxidation in vitro by the Fenton reaction by adding FeSO4 and hydrogen peroxide solutions. The content of nonprotein sulfhydryl groups was determined after protein precipitation spectrophotometrically with 5.5'-dithio-bis-2-nitrobenzoic acid. Results. The intensity of spontaneous (basic) oxidation doesn't have significant differences between the indicators of breast and cow's milk. Significant differences were established in the content of carbonyl derivatives of amino acid residues of human and cow's milk proteins during metal-catalyzed oxidation. Incubation with iron ions caused 1.5-2.5 fold more formation of both aldehyde and ketone derivatives of cow's milk proteins, recorded in the visible and ultraviolet spectrum. In cow's milk during spontaneous oxidation and induction of oxidation by a metal, the percentage of aldehyde-dinitrophenylhydrazones was lower than in breast milk and, conversely, the proportion of ketone-dinitrophenylhydrazones, late markers of oxidative degradation of proteins, was significantly higher. The content of non-protein sulfhydryl groups in cow's milk was 2 times less than in fresh human milk. A significant excessive content of aldehyde-dinitrophenylhydrazones (2 times) and ketone-dinitrophenylhydrazones (2.6 times) undet metal-catalyzed protein oxidation of cow's milk in comparison with breast milk indicates a lower level of antioxidant reserves of cow's milk. This is confirmed by the reduced level of non-protein sulfhydryl groups. The results obtained indicate the need to improve the antioxidant status of dairy products for infant nutrition.

РЕЗУЛЬТАТЫ МОНИТОРИНГА ЗА ВИРУСОМ ГЕПАТИТА Е И АЛГОРИТМ ЕГО ЛАБОРАТОРНОГО КОНТРОЛЯ

Background. Individual cases of viral hepatitis E are recorded in the Republic of Belarus annually indicating the need for the pathogen monitoring at both the population and reservoir levels. Objective. To consolidate the monitoring data on hepatitis E virus over the period of 2018 - 2021, as well as to work out an effective algorithm for its laboratory screening. Material and methods. As part of the study, 345 samples were analyzed, including 227 human biological samples, 37 samples of biological materials of domestic pigs, 22 samples of food and 59 samples of waste water. Results. According to the results of serum diagnostics, in the group of kidney recipients (n = 29), the detection rate of IgM and IgG to hepatitis E virus was 6.9% [0.85%; 23.03%] and 17.2% [7.13%; 35, 02%] respectively; in the group of patients with pregnancy pathology (n = 44) - 6.8% [1.68%; 18.89%] and 11.4% [4.5%; 24.43%] respectively. In patients with acute hepatitis of unknown etiology (n = 26), antiviral IgM was not detected, while the frequency of antiviral IgG detection reached 7.7% [1.02%; 25.26]. In control group (blood donors, n = 53) IgM and IgG were detected in 1.9% [0.6%; 10.88%] and 5.7% [1.35%; 15.97] of those examined respectively. Hepatitis E virus RNA was detected in 8 human biological samples (3.8%) from kidney recipients. The identified hepatitis E viruses were represented by genotype GIII and belonged to a previously unidentified subgenotype (GIIIa - GIIIi). In the studied samples of biological material from pigs, as well as in samples of food and waste water, hepatitis E virus RNA was not detected. Conclusions. An algorithm for hepatitis E virus laboratory screening has been developed and tested. Its section concerning the diagnosis of viral hepatitis E is set out in the Instructions for use "Algorithm for laboratory diagnosis of viral hepatitis E" (No. 148-1220 from January 28, 2021).

Detection of circulating tumour cells in colorectal cancer: Emerging techniques and clinical implications.

Despite several advances in oncological management of colorectal cancer, morbidity and mortality are still high and devastating. The diagnostic evaluation by endoscopy is cumbersome, which is uncomfortable to many. Because of the intra- and inter-tumour heterogeneity and changing tumour dynamics, which is continuous in nature, the diagnostic biopsy and assessment of the pathological sample are difficult and also not adequate. Late manifestation of the disease and delayed diagnosis may lead to relapse or metastases. One of the keys to improving the outcome is early detection of cancer, ease of technology to detect with uniformity, and its therapeutic implications, which are yet to come. "Liquid biopsy" is currently the most recent area of interest in oncology, which may provide important tools regarding the characterization of the primary tumour and its metastasis as cancer cells shed into the bloodstream even at the early stages of the disease. By using this approach, clinicians may be able to find out information about the tumour at a given time. Any of the following three types of sampling of biological material can be used in the "liquid biopsy". These are circulating tumour cells (CTCs), circulating tumour DNA, and exosomes. The most commonly studied amongst the three is CTCs. CTCs with their different applications and prognostic value has been found useful in colorectal cancer detection and therapeutics. In this review, we will discuss various markers for CTCs, the core tools/techniques for detection, and also important findings of clinical studies in colorectal cancer and its clinical implications.

THE EFFECTIVENESS OF THE USE OF VARIOUS ISSR-MARKERS IN THE STUDY OF HORSES

The article presents the results of molecular genetic studies performed on samples of biological material from ancient horses (DNA was isolated from fossils of Pleistocene horse and tarpan) and three aboriginal breeds of modern horses (Polish horse, Hutsul breed, Arabian breed). The research was conducted in the laboratory of genetics IRGT. M.V.Zubets NAAS. Purpose of research. This work was carried out to compare the effectiveness of each of the ISSR-markers and then select the optimal combination in the study of polymorphism of the genetic structure of horses To study the DNA polymorphism of horses on ISSR markers, we used eight primers that are considered the most informative (AG)8CA, (AG)9C, (GA)6CC, (GA)9C, (AG)8CG, (GAG)6C, (ACC)6G, (CTC)6C.
 Research methods. DNA from the blood was isolated using a set of reagents "DNA sorb B". From fossil remains of horses, DNA was isolated by an optimized method using proteinase K and dithiothreitol. The PCR mixture contained: 1 μl of buffer for Tag polymerase, 1 μl of a mixture of triphosphates ("Amplisens", RF), 0.8 μl of the appropriate primer, 0.2 μl of DNA polymerase ("Fermentas", Lithuania), water for PCR 3 μl. Genomic DNA was added in an amount of 4 μl. The total volume of the DNA mixture was 10 μl. Amplification was performed on a programmed four-channel thermocycle "Tertsyk" ("DNA technology", Russia). The amplification program included primary denaturation (95°C, 2 min); 30 cycles of denaturation (95°C, 30 s), hybridization of primers (54–64°C, 30 s) and elongation (72°C, 1 min), finishing elongation (72°C, 5 min).
 To more accurately estimate the lengths of the detected amplification fragments, a universal scale was used, where the gradation of DNA fragments by molecular weights was used. Depending on the zone ("heavy", "medium" and "light" fragments), a certain step from 10 to 200 bp was used. As a result, 38 zones with a fixed interval were identified, which allow to accurately determine the molecular weight for amplification products of different lengths and standardize the results of this study.
 Obtained results and conclusions. In the study of the obtained spectra of amplification products, we found that the largest number of loci was obtained by using as primers the sequences (GA) 6CC and (GAG) 6C – 9 and 8 loci. At the same time, primers (AG) 8CA – 0.27, (AG) 9C – 0.21 and (ACC) 6G – 0.21 were the most polymorphic in terms of PIC. It should be noted that when using primer (GA) 6CC in the study of genetic polymorphism of horses was obtained a significant range of amplification products – 9 loci, with a polymorphism index of 0.16, which allows it to be used quite effectively for research. Using the sequence (AG) 8CG was characterized by the lowest PIC index and was 0.02. As a result, we found that the most effective for the detection of polymorphism in the PIC index in horses was the use of primers sequences (AG) 8CA, (AG) 9C, and (ACC) 6G. To obtain the largest range of amplification fragments in horses by ISSR-PCR, the most effective was the use of the sequence (GA) 6CC and (GAG) 6C.

Techniques of blood sampling for detection of African swine fever virus in wild boar and domestic pigs in the field conditions

It is thought that due to the high virulence of the African swine fever virus its circulation in the Russian Federation is accompanied by a low seroprevalence. However taking into account a long-term ASF unfavourable situation, the introduction of the virus into the wild boar population, and the occurrence of attenuated viral variants, the significance of serological testing aimed at the detection of viral antibodies is increasing. To collect field samples of biological material from animals for molecular genetic, virological, and serological tests, filter paper, as well as swabs, can be used. The specificity and sensitivity of enzyme-linked immunosorbent assay when testing blood absorbed by filter paper are worse than those shown when testing sera, but they allow effective detection of African swine fever virus antibodies. It was demonstrated that blood absorbed on filter paper can be used for the immunoblot analysis, but the optimum performance could be achieved when the immunoperoxidase technique in combination with samples, taken by swabs was used. When comparing results of enzyme-linked immunosorbent assay performed on sera collected from domestic pigs (infected with ASFV isolates Antonovo 07/14 and Sobinka 07/15), and blood from ear veins absorbed on filter paper the sensitivity was 88.9%, specificity – 90.6%. However, the use of the immunoperoxidase technique for testing blood from swabs showed 100% coincidence with ELISA, while testing of sera with immunoperoxidase technique was superior to ELISA in sensitivity. This means blood sampling using swabs may be recommended for tests after proper validation. This technique can be especially useful for collecting data about infected wild boars because effective eradication strategies are impossible without such data.

Determination of the causes of allergic reactions to tuberculin in cattle

The article presents the results of cattle examining in four free from tuberculosis livestock farms during 2020–2021. Samples of biological material were collected and studied in the Laboratory for tuberculosis study of the NSC “IECVM”. The causes of allergic reactions to mycobacterial allergens were established by a comprehensive method. The study was aimed to conduct epizootological monitoring and to determine the causes of positive tuberculin skin tests in cattle in four free from tuberculosis farms. These farms were located in different regions of Ukraine. Epizootological, clinical, allergical, anatomopathological, bacteriological and biological methods were used including a pathological examination of biological material samples (lymph nodes and internal organs), Ziehl–Nielsen staining while bacterioscopy. Samples of biological material were preliminary treated by A. P. Alikaeva’s method and 0.9% solution of cetylpyridinium chloride and inoculated on selective nutrient media for mycobacteria cultivation. As the result of conducted study seven cultures of nontuberculous mycobacteria were isolated from samples of biological material from three cattle herds. It was found that these isolates were represented by four mycobacterial species. There were M. fortuitum, M. phlei, M. smegmatis and M. scrofulaceum. In addition, two cultures of M. avium subsp. paratuberculosis were isolated from one cattle herd. Short-term sensitization to tuberculin for mammals in cattle was caused by atypical mycobacteria in three farms. There were four mycobacteria species; M. smegmatis, M. phlei, M. fortuitum and M. scrofulaceum which persists in the body of animals and does not cause the development of an infectious tuberculosis process. Mycobacterium avium subsp. paratuberculosis (MAP) causes the latent form of an infectious process in the body of cattle and sensitization to tuberculin, as well as pathological lesions in the small intestine. One-month-old rabbits susceptible to MAP can be used as an experimental model for determination of biological properties of epizootic cultures and diagnosis of paratuberculous enteritis. Herds of cattle in which sensitization is triggering by atypical mycobacteria should be considered as free from tuberculosis. Control of welfare and differentiation of nonspecific reactions to tuberculin should be carried out using a simultaneous test with PPD tuberculin for mammals and the allergen from atypical mycobacteria. The study of cattle with a suspicion of paratuberculous enteritis should be carried out by complex method using allergical, serological (CFT, ELISA), pathological, bacteriological and molecular-genetic research methods, as well as using a biological test on one month old rabbits